Endothelium plays a major role in the regulation of primary hemostasis, fibrinolysis, and vasomotor tone. These cells synthesize von Willerbrand factor (vWF), tissue-type plasminogen activator (tPA), and plasminogen-activator inhibitor (PAI), and vWF and tPA often seem to be released together in vivo in response to such stimuli as exercise, venous occlusion, or vasopressin (AVP). The goal of these investigations is to better understand the endothelial synthesis and release of vWF, tPA and PSI and the endothelial response to AVP. Experiments will be performed on cultured human endothelial cells obtained from umbilical veins, adult arteries and veins, and adipose tissue microvasculature. Intracellular vWF Structure and Release. The storage organelle for vWF (Weibel-Palade body) will be purified by colloidal silica density gradient centrifugation. The vWF multimer distribution will be evaluated by immunoblotting of agarose gels and compared to that of plasma vWF. Other proteins contained in the same secretory granule will be characterized; in particular, whether tPA is the same secretory organelle with vWF will be determined. Streptokinase (SK) therapy is associated with a rapid increase in plasma vWF. The exact stimulus for release will be determined by testing components of the SK-induced fibrinolytic state (ie SK, plasmin, and fibrinogen degradation products) for the ability to induce vWF release from cultured cells. Dextran on the other hand lowers vWF level. To evaluate the mechanism, the effect of dextran on endothelial protein synthesis, vWF synthesis, vWF release, and cell activation by agonist (ie thrombin) will be examined. Endothelial Regulation of Fibrinolysis. PAI will be purified from conditioned media of human endothelial cells and amino acid sequence obtained. Because PAI levels are very high during pregnancy, effect of steroid hormones on the rate of synthesis will be determined. The inactivation of PAI by activated protein C will be examined to determine whether PAI is proteolytically degraded. Endothelial vasopressin receptors. AVP stimulates release of vWF, tPA, platelet activating factor and "endothelium-derived relaxing factor." 3H-AVP binding to endothelial cells will be examined to determine whether specific, high-affinity binding sites exist and in what types of vessels, whether they are V1 or V2 receptors, and whether cytosolic calcium or cAMP second messengers are generated.